Summary Statement and Abstract Stem cell technology is revolutionizing regenerative medicine and cell-based therapies. One of the promising applications of this technology is stem cell-derived platelet. Platelets are in high demand for a number of medical treatments but general or matching sources may be limited for various reasons. A continuous, donor-independent, and universally compatible supply of platelets are thus highly desirable. We have previously developed a unique mRNA-based technology of reprogramming somatic cells into iPSCs (induced pluripotent stem cells) with over 80% efficiency in a short period of just 7~8 days. Together with our collaborators, we have recently published a feeder-free protocol to differentiate iPSCs to megakaryocytes (MKs) of ~80% purity and to universal, i.e. HLA-negative, platelets with remarkable similarity and comparable purity to human platelets. We propose the current phase I SBIR project to overcome the two remaining barriers in the translation of our technology into commercially viable clinical use. One is the prohibitive cost of manufacturing iPSC-derived platelets due to the high cost of GMP-grade recombinant growth factors and cytokines, amounting to $87,252/dose. The second barrier is the lack of automated large-scale manufacturing of the stem cells, differentiated cells, and platelets. We propose three aims. The first aim will utilize our strength in mRNA-based protein expression to manufacture growth factors/cytokines in conditioned cell media at a fraction of the cost of GMP-level recombinant growth factors/cytokines. These growth factors/cytokines will be tagged with fluorescent proteins in Phase I so that we can monitor their concentrations during the differentiation culturing from iPSCs to platelets. The second aim will optimize and improve the efficiency and yields of both megakaryocyte (MK) differentiation and platelet release from MKs. To further reduce the production cost, we will also titrate down the concentrations of growth factors/cytokines needed for the manufacturing of platelets. The third aim will implement the optimizations from the first 2 aims in a large-scale culture to manufacture platelets in quantity equivalent to at least two doses for human transfusion. Through both increasing the yields of platelets and reducing the cost of supplying growth factors/cytokines, our goal is to bring the per dose cost of iPSC-derived platelets low enough to start planning clinical trials.